1. Field of the Invention
This invention in general relates to optical scanning systems and, in particular, to scanning systems such as fluorescent microarray readers, DNA micro-array readers, or xe2x80x9cbio-chipxe2x80x9d readers, in which excitation radiation of various wavelengths are used to produce fluorescence in a scanned sample.
2. Description of the Prior Art
The use of excitation radiation to produce fluorescence in a scanned sample is known. U.S. Pat. No. 5,381,224 issued to Dixon et al. discloses scanning optical imaging systems for macroscopic specimens, the system allowing both confocal and non-confocal imaging to be performed in reflected light. Fluorescent imagers are used to acquire data in experiments that utilize fluorescent labels to identify the state of a sample being tested. In some cases the presence of or lack of fluors in the sample determines the experimental result. In other cases the density of the fluors, a function of the intensity of the radiation emitted from the sample, is the measurement of interest and the experimental result can be inferred by measuring the intensity of the detected radiation.
An example of a process that uses fluorescent labels is the microarray. A microarray is a set of experiments involving DNA (or RNA) bound to a glass substrate. Reference or xe2x80x9ctargetxe2x80x9d DNA is spotted onto a glass substratexe2x80x94typically a one- by three-inch glass microscope slidexe2x80x94where it chemically binds to the surface. Each spot, or sample, of DNA constitutes a separate experiment. xe2x80x9cProbexe2x80x9d DNA or RNA which has been labeled with a fluorophor is then introduced to the surface of the slide and is allowed to hybridize with the target DNA. Excess probe DNA that does not bind with target DNA is removed from the surface of the slide in a subsequent washing process.
The experiment allows the binding affinity between the probe and target DNA to be measured to determine the likeness of their molecular structures; complementary molecules have a much greater probability of binding than unrelated molecules. The probe DNA is labeled with fluorescent labels that emit a range of radiation energy centered about and including a wavelength xcexemission when excited by an external radiation source of a shorter wavelength xcexexcitation. The brightness of emitted radiation is a function of the fluor density in the illuminated sample. Because the fluor density is a function of the binding affinity or likeness of the probe molecule to the target molecule, the brightness of each sample can be mapped as to the degree of similarity between the probe DNA and the target DNA present. On a typical microarray up to tens of thousands of experiments can be performed simultaneously on the probe DNA, allowing for a detailed characterization of complex molecules.
A scanning fluorescent imager divides the area of interest into a set of discrete image elements referred to as pixels. Each pixel is independently addressed and measured for the presence of fluors. The fluors are excited by an incident excitation beam and a portion of the resulting emitted fluorescence radiation is collected and measured by detection apparatus. Each measurement results in a data point that represents the relative fluor density of the measured pixel. The pixel data is then reconstructed to create a quantified representation of the area scanned.
In a scanning microscope, each pixel is illuminated independently while it is being addressed. The light source is typically a single-wavelength laser device focused down to form a spot of the desired size. Radiation is emitted by the fluors in an outward, hemispherical transmission pattern. A portion of this emitted radiation is collected by beam collection optics and directed to the detection apparatus. Additional radiation collected is radiation from the incident excitation beam which is reflected or scattered by the surface of the sample. The imager optics must discriminate between the two radiation wavelengths by rejecting the excitation radiation and passing the fluorescent radiation. Optical filtering components, such as dichroic and band pass filters, provide the discrimination in conventional systems.
Laser fluorescence micro-array scanners incorporate the ability to deliver multiple laser excitation wavelengths so that fluorescence data can be obtained from the sample at two or more emission wavelengths by detecting two or more fluorescent dyes. Such a unique excitation and emission wavelength pair is typically referred to as a xe2x80x9cChannelxe2x80x9d. Many DNA micro-array samples utilize a two-wavelength scanning method, where the results of one wavelength scan are used as control values and the results of the other wavelength scan represent the desired experimental result, as in Differential Gene Expression. As the market and application mature, and a larger variety of suitable dyes become available, the demand for alternative excitation wavelengths and emission bands will increase.
Most scanning confocal microscopes employ a dichroic or multichroic beam splitter for color separation between the excitation radiation wavelength xcexexcitation and the emission radiation wavelength xcexemission. U.S. Pat. No. 5,672,880 issued to Kain, for example, discloses a fluorescence imaging system in which fluorescent light emitted by a sample is collected by an objective and passed through a dichroic filter placed along the optical axis between a laser and the objective to direct the fluorescent light onto a photo-detector. Dichroic beam splitters are fabricated using a vacuum deposition process in which inorganic crystalline materials having varying indices of optical refraction are deposited in layers onto optical substrates to create optical filters with specific band-pass and/or band-reject characteristics.
In practice, an optical scanning system may operate utilizing five or more single-wavelength radiation devices producing ten or more unique, but variable, emission bands. These operating parameters impose a specification requirement that the component multichroic optical element be designed so as to reflect all five wavelengths and pass the emission wavelengths. A first drawback to this approach is that such a beam splitter specification may be quite difficult to achieve in practice. Moreover, future improvements and developments in optical scanning systems may necessitate that the systems operate with even more excitation and emission wavelengths, requiring a multichroic beam splitter having an even more demanding specification requirements.
Another drawback of conventional optical scanning systems is the design complexity incurred by the use of single-wavelength radiation devices. By utilizing one or more multi-wavelength radiation devices, with an appropriate wavelength selection device, a more compact, robust optical scanning system can be achieved.
While the art describes a variety of imaging systems for optical scanning, there remains a need for improvements that offer advantages and capabilities not found in presently available scanners, and it is a primary object of this invention to provide such improvements.
It is another object of the present invention to provide an imaging system which can image a sample utilizing two or more different wavelengths of excitation radiation on a single microarray sample.
It is yet another object of the present invention to provide an imaging system which utilizes excitation devices producing two or more wavelengths of radiation.
It is further an object of the present invention to provide an optical scanning system which can be adapted for use with newly-available fluors without incurring the need to reconfigure the imaging system.
Other objects of the invention will be obvious, in part, and, in part, will become apparent when reading the detailed description to follow.
The present invention discloses an imaging system comprising a radiation device providing excitation radiation of different wavelengths, an objective lens for focusing the excitation radiation onto a sample to produce fluorescent emission, and a mirror, configured as a geometric beam splitter, disposed in the transmission path of the emission radiation and excitation radiation subsequent to reflection from the sample and collimation by the lens, the mirror reflecting one of the collimated excitation and emission radiation such that the emission is directed to a detector and the collimated excitation is directed away from the detector. Other features of the invention will be readily apparent when the following detailed description is read in connection with the drawings.